Fluorescent Nucleoside Analogs that Mimic Naturally Occurring Nucleosides

Background: The need to monitor, track, and quantify natural biological processes has engendered many strategies for fluorescence labeling of nucleic acids. However, fluorescent nucleoside analogs typically trade-off structural and chemical integrity for high-emission quantum efficiency and long-emission wavelength. The challenge has been to design traceable nucleoside analogs that are functional equivalents of their natural counterparts.

Technology Description : The invention describes synthesis, compositions, and methods related to the use of fluorescent nucleoside analogs as probes for nucleic-acid structure, dynamics, and function, as well as sequence and lesion analysis. These fluorescent nucleoside analogs are similar in structure to naturally occurring nucleoside bases with regard to shape, size, hybridization, and recognition properties. They also have been designed toward favorable photophysical characteristics, including emission spectra in the longer wavelengths (towards or in the visible range) and a red-shifted absorption spectrum that minimally overlaps the absorption spectrum of naturally occurring nucleosides. The exquisite sensitivity of emission spectra to the local microenvironment confers utility as probes for dynamic and recognition processes.

Advantages: The technical advances are complemented by commercial advantages including:

  • A scalable, simple synthesis using readily available precursors.
  • Easy configuration into proprietary, end-user “kits” and compatibility with solid-phase, automated synthesizers.
  • Ability to directly incorporate via enzymes of interest–as validated for in vitro transcription reactions using T7RNA polymerase.
  • Suitability for sensitive, real-time assays.

In short, these probes should prove invaluable for studying the structure and dynamics of nucleic acids and their complexes with small molecules or proteins. This includes the study of nucleic-acid modifying enzymes that play crucial roles in development, genetic diseases, and cancers as well as the identification of novel antibiotics targeted at the bacterial ribosome.

Patent Information: Patent pending, please refer to 20080261823.

Related Materials:

  • Srivatsan, S.G. and Y. Tor, Enzymatic Incorporation of Emissive Pyrimidine Ribonucleotides Chem. Asian J. 2009, In Press.
  • Greco, N. et. al., An Emissive C Analog Distinguishes between G, 8-oxoG, and T Org. Lett., Publication Date (Web): February 5, 2009; DOI: 10.1021/ol802656n.
  • Srivatsan, S.G. et. al. A Highly Emissive Fluorescent Nucleoside that Signals the Activity of Toxic Ribosome-Inactivating Proteins. Angew. Chem. Int. Ed. 2008, 47, 6661-6665.
  • Srivatsan, S.G. and Y. Tor, Synthesis and enzymatic incorporation of a fluorescent pyrimidine ribonucleotide. Nature Protocols, 2007, 2, 1547-1555.
  • Tor, Y. Fluorescent Nucleoside Analogues: Synthesis, Properties and Applications (Editorial) Tetrahedron, 2007, 63, 3425-3426.
  • Srivatsan, S.G. and Y. Tor, Using an Emissive Uridine Analogue for Assembling Fluorescent HIV-1 TAR Constructs. Tetrahedron 2007, 63, 3601-3607.
  • Greco, N. and Y. Tor, Furan Decorated Nucleoside Analogues as Fluorescent Probes: Synthesis, Photo-physical Evaluation, and Site-Specific Incorporation. Tetrahedron 2007, 63, 3515-3527
  • Greco, N.; Tor, Y. Synthesis and site-specific incorporation of a simple fluorescent pyrimidine. Nature Protocols 2007, 2, 305-316.
  • Greco, N.; Tor, Y., Fluorescent Pyrimidine Ribonucleotide: Synthesis, Enzymatic Incorporation, and Utilization. J. Am. Chem. Soc. 2007, 129, 2044-2053.
  • Greco, N.; Tor, Y., Simple Fluorescent Pyrimidine Analogues Detect the Presence of DNA Abasic Sites. J. Am. Chem. Soc. 2005, 127, 10784-10785.

Keywords: kits, nucleic acids, dynamics, DNA–protein, interaction, RNA, small molecule, genetic, cancer, TAR, antibiotic, analogs, probes, sequence, lesion analysis, heterocycles, photophysical, hybridization, spectral, spectrum, absorption, fluorescence, labels, emission, fluorophore, chromophore, genomics, molecular biology, bioassay, detection, microarray, reporter, labeling

Case Number: SD2005-116

Inquiries To:  invent@ucsd.edu